TWEEZER PEPTIDE MIMICS OF THE ESTROGEN RECEPTOR FOR DETERMINATION OF ENDOCRINE DISRUPTOR CHEMICALS Rosa Romeralo-Tapia Supervisor: Prof. Dr. Johan Van der Eycken Laboratory for Organic and Bioorganic Synthesis Thesis submitted to obtain the degree of Doctor in Sciences: Chemistry 13 October 2011
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TWEEZER PEPTIDE MIMICS OF THE ESTROGEN
RECEPTOR FOR DETERMINATION OF
ENDOCRINE DISRUPTOR CHEMICALS
Rosa Romeralo-Tapia
Supervisor: Prof. Dr. Johan Van der EyckenLaboratory for Organic and Bioorganic SynthesisThesis submitted to obtain the degree of
Doctor in Sciences: Chemistry
13 October 2011
• Challenges in endocrine disrupting chemicals research: EST-SENDICHEM project
2/75
Overview
• Challenges in endocrine disrupting chemicals research: EST-SENDICHEM project
• Design artificial receptor
3/75
Overview
• Challenges in endocrine disrupting chemicals research: EST-SENDICHEM project
• Design artificial receptor
• Synthesis type A, B and C tweezer peptide mimics
4/75
Overview
• Challenges in endocrine disrupting chemicals research: EST-SENDICHEM project
• Design artificial receptor
• Synthesis type A, B and C tweezer peptide mimics
• Structure analysis by NMR spectroscopy
5/75
Overview
• Challenges in endocrine disrupting chemicals research: EST-SENDICHEM project
• Design artificial receptor
• Synthesis type A, B and C tweezer peptide mimics
• Structure analysis by NMR spectroscopy
• Screening tweezer peptide mimics by ACE6/75
Overview
ENDOCRINE DISRUPTING CHEMICALS
(EDCs)
“Endocrine disruptors are defined as exogenous substances or mixtures that alter the function(s) of the endocrine system and
consequently cause adverse health effects in intact organisms, or their progeny or (sub) population”*
Definition of EDCs
NATURAL and SYNTHETIC HORMONES
COOLANTS, PESTICIDES PLASTICISERS
HO
OH
OH
ClCl
ClCl
Cl
OO
O
O
ClCl
HO
OH
Cl
Cl Cl
Estradiol (E2) Polychlorinated bisphenols (PCBs)
Bisphenol-A (BPA)
Ethinylestradiol (EE) 1,1,1-Trichloro-2,2-bis- (4'-chlorophenyl)ethane (DDT)
Di-n-butylphthalate (DBP)
HO
*Definition given by International Programme on Chemical Safety
8/75
http://www.chemtrust.org.uk
ClCl
Cl
Cl Cl
ClCl
ClCl
Cl
DDT
N
N
N
NH
Cl
NH
AtrazinePCBs
Effects on wild life
DDT spil in Florida LakeReproduction-related
abnormalities
DDT and PCB in fat Greenland
Premature death
Intersex characteristics
Atrazine exposure ??
9/75
Challenges in EDC Research
• Establish cause-effect
relationships
• Obtain reliable exposure data
• Identify which chemicals cause
endocrine disruption10/75
Challenges in EDC Research
• Establish cause-effect
relationships
• Obtain reliable exposure data
• Identify which chemicals cause
endocrine disruption
• Low concentrations (below
ng/L)
11/75
NEW SORBENT TO SELECTIVELY
RETAiN EDCs FROM WATER
EDC-CONTAMINATED WATER
Solid phase affinity extraction
Prof. dr. Annemieke Madder (Coordinator)
Organic and Biomimetic Chemistry
Prof. dr. P. SandraSeparation Sciences
Prof. dr. F. Du Prez Polymer Chemistry
Prof. dr. J. Van der Eycken Organic and Bioorganic Synthesis
Prof. dr. J. MartinsNMR & Structure Analysis
12/75
Mode of action of EDCs
A) Hormone-related mechanismsHormone synthesis (1)Hormone transport (2, 3, 11) Hormone metabolism (4)
www.medscape.com Source: Expert Rev Of Obstet Gynacol 2008
13/75
Mode of action of EDCs
B) Receptor-mediated mechanisms
A) Hormone-related mechanismsHormone synthesis (1)Hormone transport (2, 3, 11) Hormone metabolism (4)
www.medscape.com Source: Expert Rev Of Obstet Gynacol 2008
14/75
http://whqlibdoc.who.int
B) Receptor mediated EDCs GENISTEIN (natural hormone)
ETHINYLESTRADIOL, DIETHYLSTILBESTROL (synthetic
hormones)
DDT (pesticide), BISPHENOL-A (plasticiser)
• Agonist: mimicking the natural hormone ER
recognizes
• Antagonist: blocking the hormone receptor positions EDCs as
ligands
Mode of action of EDCs
Natural hormone
EDC
ER
ER Estrogen Receptor
15/75
NEW SORBENT TO SELECTIVELY
RETAiN EDCs FROM WATER(based on receptor-
mediated mechanism)
EDC-CONTAMINATED WATER
Solid phase affinity extraction
Van der Plas SE et al. Eur. J. Org. Chem. 2009, 11 (Sp. Iss. SI), 1796-1805Figaroli S, Madder A. Tetrahedron, 2010, 66, 6912-6918
Solid support (Polymer) Molecule with affinity to EDCs:MIMIC OF
ESTROGEN RECEPTOR
16/75
MODEL DESIGNS FOR MIMICS OF
HORMONE BINDING DOMAIN ESTROGEN RECEPTOR
(HBD-hER)
Design HBD-hER mimic
hER: 595 AA, 7 active sites
C terminal: HBD 297-595 (~300 AA)Brzozowski et al. Nature 1997, 389, 753-758
Tannenbaum et al. Proc. Natl. Acad. Sci. 1998, 95, 5998-6003
ScaffoldScaffoldHormone binding domain
Artificial receptorHormone Binding ActivityFew selected Amino acids
18/75
ScaffoldScaffoldHormone binding domain
ANCHORING AMINO ACIDS EDCs-ER COMPLEXES
Design HBD-hER mimic
19/75
Histidine 524 (H11), Arginine 394 (H5), Glutamic acid 353 (H3)
hER-E2Hydrophilic interactions
Design HBD-hER mimicAmino acids
OH
HOEstradiol (E2)
Tanenbaum D.M et al. Proc. Natl. Acad. Sci. USA 1998, 95, 5998-6003
20/75
A-B rings: Ala 350 (H3), Leu 387 (H5), Leu 391 (H5), Phe 404 (βturn), Met 388 (H5) D ring: Leu 525 (H11), Ile 424 (H7), Gly 521 (H11)
hER-E2 Hydrophobic residues
Design HBD-hER mimicAmino acids
Tanenbaum D.M et al. Proc. Natl. Acad. Sci. USA 1998, 95, 5998-6003
21/75
DESHydrophilic
contactsGlu 353 Arg 394 His 524
Hydrophobic
contacts
Ala 350
Leu 386Leu 387Phe 404Met 421Leu 525
RaloxifeneHydrophilic
contactsGlu 353
Arg 394
His 524
Hydrophobic
contactsAla 350
Leu 354Leu 387Phe 404Met 421Leu 525
E2Hydrophilic
contactsGlu 353 Arg 394
His 524
Hydrophobic
contactsAla 350
Leu 387Met 388Leu 391Phe 404Ile 424Gly 521Leu 525
Design HBD-hER mimicAmino acids
GluArgHisAlaLeuMetPhe
Data obtained from Protein Data Bank http:// www.pbd.orgDES: diethylstilbestrol E2: 17Beta-estradiol
22/75
.H1H2..MGLLTNLADREL..H4..LEILMIGLVWR.. βturn ..H6H7H8H9H10..GMEHL.. Helix 3 Helix 5 Helix 11 H12..C terminal
His 524 (H11) Arg 394 (H5)Glu 353 (H3)
Ala 350 (H3)Leu 387 (H5)Leu 391 (H5)Phe 404 (βturn) Met 388 (H5) Leu 525 (H11) Ile 424 (H7)Gly 521 (H11)
N
C
H11H3
H5
H7
Design HBD-hER mimicTertiary structure
N C
C
23/75
CHEMICAL PLATFORM
ScaffoldScaffoldHormone binding domain
Design HBD-hER mimic
24/75
Pg2HN
NHPg1
O
OH
• CO2H to be attached on solid support – Solid phase synthesis
• Aromatic ring (planar, Π-Π interactions)
• Two amines orthogonally protected
• P1-P2: Peptide strands
(building blocks= Ala, Leu, Phe, Met, Glu, Arg, His)
Design HBD-hER mimicDipodal scaffold
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Pg2HN
NHPg1
O
OH
• CO2H to be attached on solid support – Solid phase synthesis
• Aromatic ring (planar, Π-Π interactions)
• Two amines orthogonally protected
• P1-P2: Peptide strands
(building blocks= Ala, Leu, Phe, Met, Glu, Arg, His)
Design HBD-hER mimicDipodal scaffold
26/75
Pg2HN
NHPg1
O
OH
• CO2H to be attached on solid support – Solid phase synthesis
• Aromatic ring (planar, Π-Π interactions)
• Two amines orthogonally protected (Pg= protective group)
• P1-P2: Peptide strands
(building blocks= Ala, Leu, Phe, Met, Glu, Arg, His)
Design HBD-hER mimicDipodal scaffold
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P2HN
NHP1
O
OH
• CO2H to be attached on solid support – Solid phase synthesis
• Aromatic ring (planar, Π-Π interactions)
• Two amines orthogonally protected
• P1-P2: Peptide strands
(building blocks= Ala, Leu, Phe, Met, Glu, Arg, His)
Design HBD-hER mimicDipodal scaffold
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SOLID-PHASE CHEMISTRY
FOR SYNTHESIS OF MODEL PEPTIDE MIMICS
O
O
NO2
NHFmoc
HO
O
NO2
NHFmocWang Resin
Type ALinear SPPS
V.1IV.1
HO
O
NH
O
HN
NH
NH
HN
NH
NH
HN
O
O
O
O
O
HN
NH
O
O
R2
R1
R4
R5
R3
R6
O
O
Wang resinMSNT, MeIm
DMF/ CH2Cl2
NH
NH
HO
O
O NNH
N
HN
O
OHN
O
OH
ON N R1
R2
O
NH
R3
O
OH
ONN R4
Type BC-SPPS
Sequental double click
Type CC-SPPS
Linear-SPPS and click
NH
NH
HO
O
O NNH
HN
NH
HN
O
OHN
O
OH
ON N R4
R3
NH2
O
R1 O
R2 O
R5
C N
C N
HO
O
NO2
NH2
Fmoc-ClNaHCO3
dioxane/H2O
Model Peptide Mimics
Synthesis IV.1: Neustadt BR et al.Tetrahedron Lett. 1998, 39, 5317-5320
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O
O
NO2
NHFmoc
HO
O
NO2
NHFmoc Wang Resin
Type ALinear SPPS
V.1IV.1
HO
O
NH
O
HN
NH
NH
HN
NH
NH
HN
O
O
O
O
O
HN
NH
O
O
R2
R1
R4
R5
R3
R6
O
O
Wang resinMSNT, MeIm
DMF/ CH2Cl2
NH
NH
HO
O
O NNH
N
HN
O
OHN
O
OH
ON N R1
R2
O
NH
R3
O
OH
ONN R4
Type BC-SPPS
Sequental double click
Type CC-SPPS
Linear-SPPS and click
NH
NH
HO
O
O NNH
HN
NH
HN
O
OHN
O
OH
ON N R4
R3
NH2
O
R1 O
R2 O
R5N C
N C
HO
O
NO2
NH2
Fmoc-ClNaHCO3
dioxane/H2O
Synthesis IV.1: Neustadt BR et al.Tetrahedron Lett. 1998, 39, 5317-5320
Model Peptide Mimics
31/75
O
O
NO2
NHFmoc
HO
O
NO2
NHFmocWang Resin
Type ALinear SPPS
V.1IV.1
HO
O
NH
O
HN
NH
NH
HN
NH
NH
HN
O
O
O
O
O
HN
NH
O
O
R2
R1
R4
R5
R3
R6
O
O
Wang resinMSNT, MeIm
DMF/ CH2Cl2
NH
NH
HO
O
O NNH
N
HN
O
OHN
O
OH
ON N R1
R2
O
NH
R3
O
OH
ONN R4
Type BC-SPPS
Sequental double click
Type CC-SPPS
Linear-SPPS and click
NH
NH
HO
O
O NNH
HN
NH
HN
O
OHN
O
OH
ON N R4
R3
NH2
O
R1 O
R2 O
R5
C N
HO
O
NO2
NH2
Fmoc-ClNaHCO3
dioxane/H2O
N C
Synthesis IV.1: Neustadt BR et al.Tetrahedron Lett. 1998, 39, 5317-5320
Model Peptide Mimics
32/75
Type A Model Peptide Mimic
based on Linear Fmoc-SPPS
33/75
O
O
NO2
NHFmoc
O
O
NO2
HN
O
NHFmoc
Wang resin
l = 1.10 mmol/g
(i) 20% piperidine/DMF(ii) Fmoc-Gly-OH, DIC, DMAP,
CH2Cl2 (x2)
Preactivation reagents
(1st coupling)
Preactivation reagents
(2nd coupling)
Loading (mmol/g)
Yield (%)
Fmoc-Gly-OH, DIC
Fmoc-Gly-OH, DIC,
HOBt
0.58 57%
Fmoc-Gly-OH, DIC,
HOBt
Fmoc-Gly-OH, DIC,
DMAP
0.90 89%
Fmoc-Gly-OH, DIC,
DMAP
Fmoc-Gly-OH, DIC,
DMAP
1.00 Quantitative
Type A model: Linear SPPS
34/75
O
O
NO2
HN
O
NH
OHN
NH
O
OHN O
O
O
NO2
HN
O
NHFmoc
1. 20% piperidine/DMF2. Fmoc-Ala-OH, HBTU, DIPEA, DMF
3. 20% piperidine/DMF4. Fmoc-Leu-OH, HBTU, DIPEA, DMF
5. 20% piperidine/DMF
6. Fmoc-Phe-OH, HBTU, DIPEA, DMF7. 20% piperidine/DMF
8. DIPEA/Ac2O/CH2Cl2 (1/1/3)
Type A model: Linear SPPS
35/75
O
O
NH2
HN
O
NH
OHN
NH
O
OHN O
O
O
NO2
HN
O
NH
OHN
NH
O
OHN O
V.2
V.3
SnCl2. 2H2O
DMF
Crude cleaved product
Crude cleaved product
min0 5 10 15 20
mAU
0
50
100
150
200
250
300
350
11.259 min
min0 5 10 15 20
mAU
0
200
400
600
800
1000
1200
DAD1 B, Sig=254,20 Ref=off (F:\07-11-06\056-1201.D)
10.449 min
min0 5 10 15 20
mAU
0
200
400
600
800
1000
1200
)
10.449 min
Type A model: Linear SPPS
36/75
Solvent A: 5mM NH4OAc/H2O Solvent B: CH3CN 0-100% B in 15 minutes (λ = 214 nm )
O
O
O
NH2
HN
O
NH
OHN
NH
O
OHN
1. Fmoc-Gly-OH, DIC, DMAP, CH2Cl2 (x 2) 2. 20% piperidine/DMF
3. Fmoc-Glu(tBu)-OH, HBTU, DIPEA, DMF (x 2) 4. 20% piperidine/DMF
Repeat 3,45. Fmoc-Ala-OH, HBTU, DIPEA, NMP
Repeat 46. DIPEA/Ac2O/CH2Cl2 (1/1/3) (x 2)
7. 95%TFA/H20
HO
O
NH
O
HN
NH
NH
HN
NH
NH
HN
O
O
O
O
O
HN
NH
O
O
OHO
O
O
Type A model: Linear SPPS
37/75
Type B and CModel Peptide Mimicsbased on Convergent
Cu-Catalyzed 1,3 Dipolar Cycloaddition Reactions
38/75
O
O
NO2
NHFmoc
HO
O
NO2
NHFmoc Wang Resin
Type ALinear SPPS
V.1IV.1
HO
O
NH
O
HN
NH
NH
HN
NH
NH
HN
OO
O
O
O
HN
NH
O
O
R2
R1
R4
R5
R3
R6
O
O
Wang resinMSNT, MeIm
DMF/ CH2Cl2
NH
NH
HO
O
O NNH
N
HN
O
OHN
O
OH
ON N R1
R2
O
NH
R3
O
OH
ONN R4
Type BC-SPPS
Sequental double click
N C
N C
Type CC-SPPS
Linear-SPPS and click
NH
NH
HO
O
O NNH
HN
NH
HN
O
OHN
O
OH
ON N R4
R3
NH2
O
R1 O
R2 O
R5N C
C N
Peptide Mimics Synthesis
39/75
Franke R et al. Tetrahedron Lett. 2005, 46, 4479-4482
CuAACCu-alkyne azide 1,3-cycloaddition
H2N
R1
NN
NCO2H
R2
H2NN
O
R1
CO2H
R21
23
4
H
H acceptor H acceptor
H
H donor H donor
AMIDE
BOND
1,4-SUBSTITUTEDTRIAZOLE
RI NG
i) N3CH2CO-TSKYREG-OH, CuI
ii) Cleavage 1
4
40/75
O
O
NO2
NHFmocO
O
NH2
NHFmoc
HO
O
HN
NHFmoc
O
HO
O
NH2
NHFmoc
SnCl2. 2H2O, DMF +
(i) 4-pentynoic acid,
DIC, CH2Cl2(ii) 95%TFA/H2O
Scaffold towards Click Approach
41/75
O
O
NO2
NHFmocO
O
NH2
NHFmoc
HO
O
HN
NHFmoc
O
HO
O
NH2
NHFmoc
HO
O
HN
NH
O
O
SnCl2. 2H2O, DMF +
(i) 4-pentynoic acid,
DIC, CH2Cl2(ii) 95%TFA/H2O
(i) 4-pentynoic acid,
DIC, DMAP, CH2Cl2(ii) 95%TFA/H2O
Scaffold towards Click Approach
42/75
O
O
NO2
NHFmocO
O
NH2
NHFmoc
HO
O
HN
NHFmoc
O
HO
O
NH2
NHFmoc
HO
O
HN
NH
O
O
SnCl2. 2H2O, DMF +
(i) 4-pentynoic acid,
DIC, CH2Cl2(ii) 95%TFA/H2O
(i) 4-pentynoic acid,
DIC, DMAP, CH2Cl2(ii) 95%TFA/H2O
HO
O
HN
NHFmoc
O(i) 4-pentynoic acid,
HBTU, DIPEA, CH2Cl2(ii) 95%TFA/H2O
Scaffold towards Click Approach
43/75
O
O
NO2
NHFmocO
O
NH2
NHFmoc
HO
O
HN
NHFmoc
O
HO
O
NH2
NHFmoc
HO
O
HN
NH
O
O
SnCl2. 2H2O, DMF +
(i) 4-pentynoic acid,
DIC, CH2Cl2(ii) 95%TFA/H2O
(i) 4-pentynoic acid,
DIC, DMAP, CH2Cl2(ii) 95%TFA/H2O
O
O
HN
NHFmoc
O(i) 4-pentynoic acid,
HBTU, DIPEA, CH2Cl2
Scaffold towards Click Approach
44/75
New scaffold
Synthesis Azidopeptides
O
ONH
ONH2 O
ONH
O HN
N3O
R1
R2 HBTU, DIPEA, DMF (x 2)
2-ClTrt.resin
R1
R2
HBTU-mediatedFmoc SPPS
AcOH/ TFE/ CH2Cl2
ON3HO
23-70% crude purity
HO
ONH
O HN
N3O
R1
R2
Azido acetic acid coupling to free amine
min0 5 10 15 20
mAU
0
200
400
600
800
\08-12-11\ -
10.5
02 10.9
41
11.5
24
min0 5 10 15 20
mAU
0
200
400
600
800
- - \
10.5
02 10.9
41
N3CH2CO-Leu-Phe
Leu-Phe
HOBt
Synthesis N3CH2CO2H:Franke R et al. Tetrahedron Lett. 2005, 46, 4479-4482
45/75
Imidazole-1-sulfonyl azide hydrochloride: Goddard-Borger ED et al. Org. Lett. 2007, 9, 3793-3800Devulder V, Backaert F, Van der Eycken J. Ghent University. 2010
O
ONH
O HN O
ONH
O HN
N3O
R1
R22-Cl Trt. resin
R1
R2
HBTU-mediatedFmoc SPPS
AcOH/ TFE/ CH2Cl2
CuSO4. 5H2O
THF/H2O
R3
O
NH2
R3N
NSO
N3
OHCl
OH
ONH
O HN
N3O
R1
R2
R3
58-76% crude purity
Synthesis AzidopeptidesDiazo transfer reaction
min0 5 10 15 20
mAU
0
200
400
600
800
1000N3Leu-Leu-Leu-Gly-Leu-Phe-OH
N3Leu-Leu-Gly-Leu-Phe-OH
Leu-Leu-Leu-Gly-Leu-Phe-OH
46/75
O
O
NO2
NHFmoc
HO
O
NO2
NHFmoc Wang Resin
Type ALinear SPPS
V.1IV.1
HO
O
NH
O
HN
NH
NH
HN
NH
NH
HN
O
O
O
O
O
HN
NH
O
O
R2
R1
R4
R5
R3
R6
O
O
Wang resinMSNT, MeIm
DMF/ CH2Cl2
NH
NH
HO
O
O NNH
N
HN
O
OHN
O
OH
ON N R1
R2
O
NH
R3
O
OH
ONN R4
Type BC-SPPS
Sequental double click
N C
N C
Type CC-SPPS
Linear-SPPS and click
NH
NH
HO
O
O NNH
HN
NH
HN
O
OHN
O
OH
ON N R4
R3
NH2
O
R1 O
R2 O
R5
Model Peptide Mimics
47/75
O
NHFmoc
O HN
O
NN N
O
HN
O
NH
O
OH
i) 20%piperidine/DMFii) 4-pentynoic acid,HBTU, DIPEA, DMF
O
NHFmoc
O HN
O
HO
ONH
O HN
N3O
CuI, Ascorbic acid, DIPEA, 2,6-lutidine/DMF
NH
NH
HO
O
O NNH
N
HN
O
OHN
O
OH
ON N
O
NH
O
OH
ONN
HO
ONH
O HN
N3O
CuI, Ascorbic acid, DIPEA, 2,6-lutidine/DMF
i)
ii) TFA,H2O (95%)
NH
NH
O
O
O NNH
O
OHN
O
OH
ON N
Type B model: Sequential Click
48/75
New scaffold
O
NHFmoc
O HN
O
NN N
O
HN
O
NH
O
OH
i) 20%piperidine/DMFii) 4-pentynoic acid,HBTU, DIPEA, DMF
O
NHFmoc
O HN
O
HO
ONH
O HN
N3O
CuI, Ascorbic acid, DIPEA, 2,6-lutidine/DMF
NH
NH
HO
O
O NNH
N
HN
O
OHN
O
OH
ON N
O
NH
O
OH
ONN
HO
ONH
O HN
N3O
CuI, Ascorbic acid, DIPEA, 2,6-lutidine/DMF
i)
ii) TFA,H2O (95%)
NH
NH
O
O
O NNH
O
OHN
O
OH
ON N
Type B model: Sequential Click
49/75
O
NHFmoc
O HN
O
NN N
O
HN
O
NH
O
OH
i) 20%piperidine/DMFii) 4-pentynoic acid,HBTU, DIPEA, DMF
O
NHFmoc
O HN
O
HO
O
NH
O HN
N3O
CuI, Ascorbic acid, DIPEA, 2,6-lutidine/DMF
NH
NH
HO
O
O NNH
N
HN
O
OHN
O
OH
ON N
O
NH
O
OH
ONN
HO
ONH
O HN
N3
O
CuI, Ascorbic acid, DIPEA, 2,6-lutidine/DMF
i)
ii) TFA,H2O (95%)
NH
NH
O
O
O NNH
O
OHN
O
OH
ON N
Type B model: Sequential Click
50/75
O
NHFmoc
O HN
O
NN N
O
HN
O
NH
O
OH
i) 20%piperidine/DMFii) 4-pentynoic acid,HBTU, DIPEA, DMF
O
NHFmoc
O HN
O
HO
ONH
O HN
N3O
CuI, Ascorbic acid, DIPEA, 2,6-lutidine/DMF
NH
NH
HO
O
O NNH
N
HN
O
OHN
O
OH
ON N
O
NH
O
OH
ONN
HO
ONH
O HN
N3O
CuI, Ascorbic acid, DIPEA, 2,6-lutidine/DMF
i)
ii) TFA/ H2O (95%)
NH
NH
O
O
O NNH
O
OHN
O
OH
ON N
Type B model: Sequential Click
51/75
NH
NH
HO
O
O NNH
N
HN
O
OHN
O
OH
ON N
O
NH
O
OH
ONN
Crude product ( 7 steps)
min0 5 10 15 20
mAU
0
200
400
600
800
1000
DAD1 A, Sig=214,20 Ref=off (F:\09-02-09\080-1201.D)
9.2
25
9.6
86
10.
149
13.
340
min0 5 10 15 20
mAU
-100
-50
0
50
100
150
200
250
300
DAD1 A, Sig=214,20 Ref=off (F:\09-07-02\093-0501.D)
10.
285
After RP-HPLC
Purity 82%
Type B model: Sequential Click
52/75Solvent A: 5mM NH4OAc/H2O Solvent B: CH3CN 0-100% B in 15 minutes (λ = 214 nm )
O
O
NO2
NHFmoc
HO
O
NO2
NHFmocWang Resin
Type ALinear SPPS
V.1IV.1
HO
O
NH
O
HN
NH
NH
HN
NH
NH
HN
O
O
O
O
O
HN
NH
O
O
R2
R1
R4
R5
R3
R6
O
O
Wang resinMSNT, MeIm
DMF/ CH2Cl2
NH
NH
HO
O
O NNH
N
HN
O
OHN
O
OH
ON N R1
R2
O
NH
R3
O
OH
ONN R4
Type BC-SPPS
Sequental double click
Type CC-SPPS
Linear-SPPS and click
NH
NH
HO
O
O NNH
HN
NH
HN
O
OHN
O
OH
ON N R4
R3
NH2
O
R1 O
R2 O
R5N C
C N
Peptide mimics synthesis
53/75
O
NHFmoc
O HN
O
Wang resin
O
O
HN
O
HN
O
NH
OHN
NH
O
O
NHFmoc
O OtBu
i) DBU/4-methylpiperidine/DMF (3/17/80)ii)Fmoc-AA-OH, HATU, DIPEA, DMF
x 4 AA
i)
CuI, ascorbic acid, DIPEA, 2.6-lutidine/DMF, 80ºC, MW
ii) DBU/4-methylpiperidine/DMF (3/17/80)iii) TIS/TFA/H2O
NH
NH
HO
O
O NNH
HN
NH
HN
O
OHN
O
OH
ON N
NH2
O
HO O
O
O
HO
ONH
O HN
N3O
Type C model: SPPS-Click
54/75
O
NHFmoc
O HN
O
Wang resin
O
O
HN
O
HN
O
NH
OHN
NH
O
O
NHFmoc
O OtBu
i) DBU/4-methylpiperidine/DMF (3/17/80)ii) Fmoc-AA-OH, HATU, DIPEA, DMF
x 4 AA
i)
CuI, ascorbic acid, DIPEA, 2.6-lutidine/DMF, 80ºC, MW
ii) DBU/4-methylpiperidine/DMF (3/17/80)iii) TIS/TFA/H2O
NH
NH
HO
O
O NNH
HN
NH
HN
O
OHN
O
OH
ON N
NH2
O
HO O
O
O
HO
ONH
O HN
N3O
Type C model: SPPS-Click
55/75
O
NHFmoc
O HN
O
Wang resin
O
O
HN
O
HN
O
NH
OHN
NH
O
O
NHFmoc
O OtBu
i) DBU/4-methylpiperidine/DMF (3/17/80)ii)Fmoc-AA-OH, HATU, DIPEA, DMF
x 4 AA
i)
CuI, ascorbic acid, DIPEA, 2.6-lutidine/DMF, 80ºC, MW (80W)
ii) DBU/4-methylpiperidine/DMF (3/17/80)iii) TIS/TFA/H2O
NH
NH
HO
O
O NNH
HN
NH
HN
O
OHN
O
OH
ON N
NH2
O
HO O
O
O
HO
ONH
O HN
N3O
Type C model: SPPS-Click
56/75
O
NHFmoc
O HN
O
Wang resin
O
O
HN
O
HN
O
NH
OHN
NH
O
O
NHFmoc
O OtBu
i) DBU/4-methylpiperidine/DMF (3/17/80)ii)Fmoc-AA-OH, HATU, DIPEA, DMF
x 4 AA
i)
CuI, ascorbic acid, DIPEA, 2.6-lutidine/DMF, 80ºC, MW
ii) DBU/4-methylpiperidine/DMF (3/17/80)iii) TIS/TFA/H2O
NH
NH
HO
O
O NNH
HN
NH
HN
O
OHN
O
OH
ON N
NH2
O
HO O
O
O
HO
ONH
O HN
N3O
Purity 80%
min0 5 10 15 20
mAU
-100
-50
0
50
100
150
200
250
300
DAD1 A, Sig=214,20 Ref=off (10-01-25\074-3301.D)
7.9
99
8.8
69
Type C model: SPPS-Click
57/75
Solvent A: 5mM NH4OAc/H2O Solvent B: CH3CN 0-100% B in 15 minutes (λ = 214 nm )
STRUCTURE ANALYSISBY
NMR SPECTROSCOPY
“The biological function of peptides and
proteins is defined by their ability to
adopt well-defined conformations that
complement those of their binding
partner […]”
Pedersen DS, Andrew Abell Eur. J. Org. Chem. 2011, 2399-2411
2D J-correlation experiment
2D-NMR spectroscopy: H-H TOCSY
59/75
NH
NH
HO
O
O NNH
N
HNO
OHN
OOH
ON N
O
NH O
OH
ONN
H
H
HH
HH
H
H
Type B peptide mimic : H-H TOCSY
700 MHz, 25°C, CD3CN/H2O
ppm
7.27.47.67.88.08.2 ppm9
8
7
6
5
4
3
2
1
Leu NH
Leu H
Leu HLeu Hγ
Leu Hδ Leu Hδ
Phe NH
Phe H
Phe H
Phe H
In collaboration with K Gheysen, Prof dr J Martins
60/75
NH
NH
HO
O
O NNH
HN
NH
HN
O
OHN
O
OH
ON N
NH2
O
HO O
O
CH3 O
H HH
H
H
H
HH
Gly1
Gly2
700 MHz, 25°C, CD3CN/H2O
Type C peptide mimic : H-H TOCSY
ppm
7.27.47.67.88.08.2 ppm9
8
7
6
5
4
3
2
1
Phe Hβ
Phe Hβ
Phe H
Phe NH
Gly1 H
Gly1 H
Gly1 NH
Gly2 H
Gly2 H
Gly2 NH
Ala NH
Ala Hβ
Ala H
In collaboration with K Haustraete, K Gheysen, Prof dr J Martins
61/75
Type A peptide mimic : H-H TOCSY
H (NH) signals
700 MHz, 25°C, CD3CN/H2O
HO
O
NH
O
HN
NH
NH
HN
NH
NH
HN
O
O
O
O
O
HN
NH
O
O
OHO
O
O
In collaboration with K Gheysen, Prof dr J Martins
62/75
2D nOe-correlation experimentCoupling through space
2D-NMR spectroscopy: H-H NOESY
63/75
.
1H-1H nOeSY (RED)600 ms mixing time, 700 MHz, 25°C, CD3CN/H2O (5/1)
HO
O
NH
O
HN
NH
NH
HN
NH
NH
HN
O
O
O
O
O
HN
NH
O
O
OHO
O
O
nOe contact
NH H alpha
N to C direction
ppm
7.27.47.67.88.08.28.48.6 ppm9
8
7
6
5
4
3
2
1
H alpha Phe
NH Leu
nOe contact
Type A peptide mimic: H-H NOESY
In collaboration with K Gheysen, Prof dr J Martins
64/75
.
NMR-monitoring H/D exchange
N
O
HN
HN
NH
HN
NH
NH
HN
O
O
O
O
O
HN
N
O
O
R1
OO
R3
R4
R2
R5
O
O N
O
DN
ND
ND
DN
ND
ND
DN
O
O
O
O
O
DN
N
O
O
R1
OO
R3
R4
R2
R5
O
O
HH
HH H H
CH3CN/D2O
CH3CN/H2O
H replaced by D: NO H BONDING
H remains: H BONDING
Different spin magnetic properties H and D nucleiH visible vs D invisible in 1H-NMR region
65/75
Type A peptide mimic: H/D exchange
BLACK: CH3CN/H2O RED: CH3CN/D2O
In collaboration with K Gheysen, Prof dr J Martins
66/75
DO
O
ND
O
DN
ND
ND
DN
ND
ND
DN
O
O
O
O
O
DN
ND
O
O
ODO
O
O
NH Phe (7.65 ppm)
7.11 ppmH orto (Phe)
7.18 ppmH meta (Phe)
1.35 ppmHβ (Leu)
nOe contacts
Type B peptide mimic: H-H NOESY
< 5Ǻ
H alpha NH
N to C direction
NH
NH
HO
O
O NNH
N
HN
O
O HN
OOH
ON N
R
O
NH O
OH
ONN
H
H H nOe contactBETWEEN STRANDS
H
H
H
600 ms mixing time 700 MHz, 25°C
CD3CN/H2O (5/1)
In collaboration with K Gheysen, Prof dr J Martins
67/75
Type C peptide mimic: H-H ROESY
ROESY: Off-resonance
H alpha NH
NH H alpha
In collaboration with K Gheysen, Prof dr J Martins
NH
NH
HO
O
O NNH
HN
NH
HN
O
OHN
O
OH
ON N
NH2
O
O
O
O OH
nOe contact non adjacent
residues (1-8)
HH N
HN
O
O
N
NN
OH
O
TURN-like-STRUCTURE
4.97 ppmtriazole-CH2-CO
7.54 ppmNH Gly2
ppm
9 8 7 6 5 4 3 2 1 0 ppm9.0
8.5
8.0
7.5
7.0
6.5
6.0
5.5
5.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
4.97 ppm“H” triazole-CH2-CO
nOe
crosspeak
7.54 ppmNH Gly2
ppm
9 8 7 6 5 4 3 2 1 0 ppm9.0
8.5
8.0
7.5
7.0
6.5
6.0
5.5
5.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
4.97 ppm“H” triazole-CH2-CO
7.54 ppmNH Gly2
ppm
9 8 7 6 5 4 3 2 1 0 ppm9.0
8.5
8.0
7.5
7.0
6.5
6.0
5.5
5.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
4.97 ppm“H” triazole-CH2-CO
7.54 ppmNH Gly2
ppm
9 8 7 6 5 4 3 2 1 0 ppm9.0
8.5
8.0
7.5
7.0
6.5
6.0
5.5
5.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
4.97 ppm“H” triazole-CH2-CO
ppm
9 8 7 6 5 4 3 2 1 0 ppm9.0
8.5
8.0
7.5
7.0
6.5
6.0
5.5
5.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
ppm
9 8 7 6 5 4 3 2 1 0 ppm9.0
8.5
8.0
7.5
7.0
6.5
6.0
5.5
5.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
ppm
9 8 7 6 5 4 3 2 1 0 ppm9.0
8.5
8.0
7.5
7.0
6.5
6.0
5.5
5.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
4.97 ppm“H” triazole-CH2-CO
nOe
crosspeak
7.94 ppmNH Gly
68/75
SCREENING OF MODEL
PEPTIDE MIMICS
AFFINITY CAPILLARY ELECTROPHORESIS
(ACE)
25 mM Tris-acetate (pH 7.4), 1 mM EDTA and 20% MeOH
Affinity Capillary Electrophoresis
17β-estradiol
Tweezer peptide mimics
_
- - EOF + +
Buffer 25 mM Tris-acetate
(pH 7.4) 1 mM EDTA 20% MeOH
Peptide mimics
17β-estradiol[E2]= 0 μM
50 μM 100 μM 200 μM
70/75
Minutes
3 4 5 6 7 8 9 10 11 12
AU
-0.002
0.000
0.002
0.004
0.006
0.008
AU
-0.002
0.000
0.002
0.004
0.006
0.008
3.49
6
6527
5.50
2
2470
5.61
9
1083
1
8.28
7
1694
10.2
88
1602
1
Minutes
3 4 5 6 7 8 9 10 11 12
AU
-0.004
-0.002
0.000
0.002
0.004
0.006
0.008
AU
-0.004
-0.002
0.000
0.002
0.004
0.006
0.0083
.13
5 6
12
3
4.7
94
38
54
.89
0 2
07
88
7.0
56
16
76
8.6
54
15
51
4
Minutes
3 4 5 6 7 8 9 10 11 12
AU
-0.002
0.000
0.002
0.004
0.006
0.008
AU
-0.002
0.000
0.002
0.004
0.006
0.008
3.4
96
6
52
7
5.5
02
2
47
05
.61
9
10
83
1
8.2
87
1
69
4
10
.28
8
16
02
1
Minutes
3 4 5 6 7 8 9 10 11 12
AU
-0.002
0.000
0.002
0.004
0.006
0.008
AU
-0.002
0.000
0.002
0.004
0.006
0.008
3.4
13
68
38
5.3
10
10
62
5.4
27
20
26
4
7.9
52
17
25
9.8
27
17
13
0
Minutes
3 4 5 6 7 8 9 10 11 12
AU
-0.004
-0.002
0.000
0.002
0.004
0.006
0.008
AU
-0.004
-0.002
0.000
0.002
0.004
0.006
0.008
3.2
96
7
10
5
5.0
88
2
00
5.1
98
3
19
09
7.6
04
1
69
7
9.3
85
1
82
00
Minutes
3 4 5 6 7 8 9 10 11 12
AU
-0.004
-0.002
0.000
0.002
0.004
0.006
0.008
AU
-0.004
-0.002
0.000
0.002
0.004
0.006
0.008
3.1
35
6
12
3
4.7
94
3
85
4.8
90
2
07
88
7.0
56
1
67
6
8.6
54
1
55
14
[E2]= 0 μM
50 μM
100 μM
200 μM
Affinity Capillary Electrophoresis
Type A PEPTIDE
MIMIC
In collaboration with V Malanchin, Dr. F Lynen, Prof dr P Sandra
71/75
EOF
Dissociation constants (Kd)
In collaboration with V Malanchin, Dr. F Lynen, Prof dr P Sandra
72/75
Strong interactions range
High affinity ligands Type A 128 μMType B 158 μMType C 124 μM
Weak interactions rangeLow affinity ligands
(μRL-μR)
Corresponding equation
LK
KL
d
dARLRRL 1
1max,
1
RRLbRRLRRL LK
max,max,
1111
RRLbRRLb
RRL KKL
max,
bRRLRRLRRL K
LL
max,max,
1
O
O
NO2
NHFmoc
HO
O
NO2
NHFmocWang Resin
V.1IV.1
Wang resinMSNT, MeIm
DMF/ CH2Cl2
NH
NH
HO
O
O NNH
N
HN
O
OHN
O
OH
ON N R1
R2
O
NH
R3
O
OH
ONN R4
Type BC-SPPS
Sequental double click
Type CC-SPPS
Linear-SPPS and click
NH
NH
HO
O
O NNH
HN
NH
HN
O
OHN
O
OH
ON N R4
R3
NH2
O
R1 O
R2 O
R5
DO
O
ND
O
DN
ND
ND
DN
ND
ND
DN
OO
O
O
O
DN
ND
O
O
ODO
O
O
General Conclusion
TYPE A MODEL PEPTIDESPPS: HBTU/DIPEA LONG REACTION TIMESNMR: CAVITY LARGER THAN 5 ǺACE: WEAK INTERACTIONS 17β-ESTRADIOL
73/75
O
O
NO2
NHFmoc
HO
O
NO2
NHFmoc Wang Resin
V.1IV.1
HO
O
NH
O
HN
NH
NH
HN
NH
NH
HN
OO
O
O
O
HN
NH
O
O
R2
R1
R4
R5
R3
R6
O
O
Wang resinMSNT, MeIm
DMF/ CH2Cl2
NH
NH
HO
O
O NNH
HN
NH
HN
O
OHN
O
OH
ON N R4
R3
NH2
O
R1 O
R2 O
R5NH
NH
HO
O
O NNH
N
HN
O
OHN
O
OH
ON N
O
NH O
OH
ONN
H
H H
H
H
TYPE B MODEL PEPTIDECuAAC SOLID PHASE: UP TO THREE DAYS REACTION TIME
NMR: INTERSTRAND H CONTACT. CAVITY NARROWER THAN 5 ǺACE: WEAK INTERACTIONS WITH 17β-ESTRADIOL
General Conclusion
74/75
O
O
NO2
NHFmoc
HO
O
NO2
NHFmocWang Resin
Type ALinear SPPS
V.1IV.1
HO
O
NH
O
HN
NH
NH
HN
NH
NH
HN
OO
O
O
O
HN
NH
O
O
R2
R1
R4
R5
R3
R6
O
O
Wang resinMSNT, MeIm
DMF/ CH2Cl2
NH
NH
HO
O
O NNH
N
HN
O
OHN
O
OH
ON N R1
R2
O
NH
R3
O
OH
ONN R4
HH N
HN
O
O
NNN
OH
ONH
NH
HO
O
O
HN
NH
HN
O NH2
O
O
O
O OH
TYPE C MODEL PEPTIDESPPS: HATU/DIPEA BETTER EFFICIENCY
CuAAC SOLID PHASE: MW ACCELERATES REACTION RATESNMR: FLEXIBILITY. TURN REGION
ACE: WEAK INTERACTIONS WITH 17β- ESTRADIOL
General Conclusion
75/75
ACKNOWLEDGEMENTS
Prof. dr. A. Madder Prof. dr. J. Martinsdr. Frederic LynenProf. dr. F. Du PrezProf. dr. P. Sandra
Prof. dr. J. Van der Eyckendr. Steven Van der Plas
dr. Els Van Hoeckdr. Talha Gökmen
Sara FigaroliVivienne Malanchin
UGent peopleJurgen CaroenJan Goeman
dr. An ClemmenKatelijne GheysenKatrien Haustraete
Tom Parveliet
UVA peopleProf. dr. Jose Martin
Marie-Curie FoundationUniversity of Ghent
TWEEZER PEPTIDE MIMICS OF THE ESTROGEN
RECEPTOR FOR DETERMINATION OF
ENDOCRINE DISRUPTOR CHEMICALS
Rosa Romeralo-Tapia
Supervisor: Prof. Dr. Johan Van der EyckenLaboratory for Organic and Bioorganic SynthesisThesis submitted to obtain the degree of
Doctor in Sciences: Chemistry
13 October 2011